Rapid impulse propagation is a vital feature of atrial and ventricular conduction system cells to maintain normal rhythmicity and coordinated contraction of the four-chambered heart. Dysregulation of these rapidly conducting cells, particularly distal His-Purkinje system (HPS) cells, have been implicated as the site of initiation of many inherited and acquired forms of cardiac arrhythmias, heart block, and sudden cardiac arrest (SCA). Our lab has shown that Neuregulin-1 (NRG1), an endocardially-derived paracrine hormone, is necessary and sufficient to convert embryonic cardiomyocytes towards a fast conduction phenotype. Furthermore, a number of studies probing the human genome have shown NRG1 is an important susceptibility gene for SCA. Despite numerous reports demonstrating that NRG1 signaling is pivotal for cardiac development and maintaining physiological heart function, the signaling mechanisms through which NRG1 functions remains unclear. In this proposal, we seek to identify transcriptional effectors downstream of NRG1 signaling that are required to confer a HPS cell fate decision. Our initial experiments using cardiac conduction system (CCS)-lacZ transgenic reporter mice and signal transduction pathway inhibitors revealed that NRG1 mediates CCS specification in the heart through the Ras-MAPK-RSK/MSK signaling cascade. In parallel, transcriptional profiling of developing and mature CCS cells compared to ventricular myocytes identified numerous enriched transcription factors of which a single MSK/RSK dependent candidate, ETV1, a member of the E-twenty-six TF family, was identified. ETV1 expression was highly restricted to rapidly conducting regions of the heart and was markedly induced in response to NRG1. The specific aims discussed in this proposal will explore the NRG1-ETV1 signaling axis and elucidate their role in regulating normal sinus rhythm. In particular, Aim 1 will address whether NRG1 though ETV1 gene regulatory networks dictate the unique expression of transcription factors, ion channels, and gap junction proteins selectively expressed in rapidly conducting atria and HPS cells. It will also determine whether there are direct ETV1 regulated genes that are important for rapid conduction. Aim 2 will test whether loss of ETV1 using global and cardiac-specific Etv1 knockout mice impact cardiac conduction parameter and specification of the HPS. These experiments will be the first to examine the link between a paracrine signaling pathway and direct downstream targets required to maintain the hierarchy of myocardial conduction velocities in the heart. We hypothesize that these studies will uncover new mechanistic insight on what governs lineage specification of specialized HPS cells versus working myocytes. In addition, the identification and characterization of these transcriptional targets could give rise to novel pathways to pursue for the development of anti-arrhythmic agents. This may bring us closer to mechanism-based therapies for arrhythmia management, improving the quality and duration of life for patients at risk of these cardiovascular disorders.